The quantitative spectrophotometric estimation of total sulfated glycosaminoglycan levels. Formation of soluble alcian blue complexes

The formation of soluble complexes between alcian blue dye and sulfated glycosaminoglycans provides the basis for the quantitative spectrophotometric estimation of the total concentration of these polysaccharides. Samples containing microgram quantities of sulfated glycosaminoglycan are mixed with a stable dye solution prepared in 15% phosphoric/2% sulfuric acids and absorbance readings at 480 nm are compared to an appropriate standard curve. The method is rapid, convenient, and reproducible. Analyses are performed under conditions in which there is no interference from the non-sulfated glycosaminoglycan hyaluronic acid, or most other anionic macromolecules. In addition, estimations are not effected by small anions or individual monosaccharides. The method has been used for the determination of the purity of commercially available preparations of hyaluronic acid and for the estimation of the sulfated glycosaminoglycan content of various biological fluids including normal human urine and the synovial fluid of individuals with rheumatoid arthritis and osteoarthritis.     

Estimation of Blood Loss in a Small Community Hospital

Three methods of blood loss estimation which are simple, accurate and cheap are: (1) weighing of sponges; (2) estimation of blood volume with Evans blue dye and (3) measurement of central venous pressure (CVP).Weighing of sponges is a valuable operating-room procedure although it has certain defects some of which are described. The Evans dye method is used chiefly in preoperative assessment when hypovolemia is suspected but serial estimations are feasible and can be performed in 45 minutes. Measurement of CVP, however, is the best single criterion of effective blood volume in relation to cardiac functional capacity and is the best guide to blood and fluid requirements. A simple “homemade” device for making serial CVP determinations, incorporating a manometer and a zero level, is described.These methods of blood loss estimation do not supersede the traditional methods of the clinical assessment of the surgical patient, but are valuable adjuncts to such assessment.     

Quantitative determination of phospholipids using the dyes Victoria blue R and B

Different phospholipids, except the choline-containing phospholipids phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin, formed complexes with the dye Victoria blue R, which selectively partitioned into the chloroform phase of chloroform/ethylene glycol/glycerol biphasic solvent system, and were quantitatively estimated at 590 nm. Considerable amounts of water, alcohols, nonlipid phosphates, neutral lipids, free fatty acids, and some detergents did not interfere with the formation of phospholipid-dye complexes. This special advantage of the method described allowed combined phospholipid extraction and estimation procedures in one test tube. Because of its high sensitivity (about 24.00 OD units/mumol of phosphatidic acid and about 10.25 OD units/mumol of other phospholipids), specificity, and simplicity, the proposed phospholipid assay appears to be very useful for rapid analyses of lipid extracts as well as TLC spots or suspensions of biological materials, as demonstrated for membranes and cells of Micrococcus lysodeikticus. The applicability of the dye Victoria blue B to the quantitative determination of phospholipids, except phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin, at 605 nm using chloroform/ethylene glycol/glycerol/water and pentane (hexane)/ethyl acetate/isopropanol/water biphasic solvent systems with similar sensitivities and of sodium dodecyl sulfate in the pentane-containing system with high sensitivity (22.96 OD units/mumol) is also shown. The adaptation of this phospholipid assay to the determination of phospholipases C and D and to the differential quantitation of choline-containing phospholipids using additional phospholipid estimation techniques is discussed.     

Colorimetric method for estimating polylysine and polyarginine

A rapid and sensitive method is described for estimating polylysine and polyarginine. The method involves the stoichiometric precipitation of these polyamino acids by excess of the anionic dye methyl orange, followed by spectrophotometric determination of unbound dye. 10 μg or less of polylysine or polyarginine can thus be estimated. The effects of various salts on the estimation are described.     

A method for the estimation of proteins in colored or turbid solutions

The many photometric methods of protein estimation give unsatisfactory results with highly colored solutions. Some plant pigment-protein complexes are not precipitable with trichloracetic acid; therefore the dye binding methods are not applicable. A rapid procedure has been devised where the protein is complexed with excess copper, the noncomplexed copper removed by an ion-exchange resin, and the copper complexed to polypeptide then estimated by atomic absorption spectrophotometry. The copper complex is proportional to the amount of protein and different proteins give the same result in the range of 0.05 to 1 mg.     

Protein assay by Coomassie brilliant blue G-250-binding method is unsuitable for plant tissues rich in phenols and phenolases

Protein estimation in crude homogenates of plant tissues rich in phenols and phenolases was carried out by the dye-binding and, with recommended cautions, by the Lowry et al. methods and the two were compared. The dye-binding method gave grossly erroneous results with a high degree of variation when the homogenizing media differed; this was not due either to the interference by the components of the homogenizing media or to any shift in the absorbance maximum. While the reduced form of the "derived" polyphenolic compounds, generated during tissue homogenization, appeared to enhance dye binding with bovine serum albumin, their influence on the protein assay directly in crude homogenates was extremely diverse. Tissue homogenization in the absence of a reducing agent results in polyquinone-protein complexes which prevent optimal dye binding, resulting in low protein values, while the endogenous phenolics in a homogenate prepared in a mixture of cysteine and NaCl appear to suppress dye-protein complex formation. It is therefore our opinion that the dye-binding method is unsuitable for protein assay in phenol- and phenolase-rich plant tissues.     

Fluorescence characteristics of DNA complexes with dyes of the bis-benzimidazole series

Fluorescence characteristics of DNA-specific dyes of bis-benzimidazole type in a wide range of pH and r = C/P were investigated. Fluorescence spectra of DNA complexes with bis-benzimidazoles have elements of a structure, which may result from a superposition of the spectra of dye molecules in different protonization group states that form different types of complexes with DNA. Experimental data do not contradict the idea of bis-benzimidazole dye binding into the minor groove of DNA. Bis-benzimide molecules in the deprotonization state have a major affinity to DNA.     

Fluorescence analysis of size distribution and mode of dye release from carboxyfluorescein-loaded vesicles: application to the study of complement-membrane interactions

We wish to report a novel method for visualizing large unilamellar vesicles loaded with a fluorescent dye and for monitoring changes in the size distribution as well as state of aggregation of such dye-loaded liposomes. In addition, we demonstrate that this method can be used to distinguish between all-or-none release of dye and graded release of dye from individual vesicles. Using this technique, we have characterized complement-mediated release of carboxyfluorescein from large unilamellar vesicles and have found that C5-8 complexes mediate a graded release of dye while C5-9 complexes cause an all-or-none release. Furthermore, complement appears to preferentially attack the medium to larger-sized vesicles in our population of large unilamellar vesicles while smaller vesicles appear to be selectively spared.     

Stoichiometries of arsenazo III-Ca complexes

The equilibrium interactions of the metallochromic indicator arsenazo III with calcium at physiological ionic strength and pH were investigated spectrophotometrically and with the aid of a calcium electrode. Evidence suggests the formation of more than one dye-calcium complex. The analysis of data obtained over a 10,000-fold range of dye concentrations concludes that at the concentrations used for in vitro biochemical studies (10--100 microM), arsenazo III absorbance changes in response to calcium binding primarily involve the formation of a complex involving two dye molecules and two calcium ions. At millimolar dye concentrations, typical of physiological calcium transient determinations in situ, a second complex involving two arsenazo III molecules and one calcium ion is additionally formed. A third complex, involving one arsenazo III molecule and one calcium ion, is formed at very low dye concentrations. The results reported here suggest that equilibrium calibration of the dye with calcium cannot be used directly to satisfactorily relate transient absorbance changes in physiological preparations to calcium concentration changes since several stoichiometrically distinct complexes with different absorbances could be formed at different rates. The results of this study do not permit the elucidation of a unique kinetic scheme of arsenazo III complexation with calcium; for this, in vitro kinetic analysis is required. Results of similar analysis of the dye interaction with magnesium are also reported, and these appear compatible with a much simpler model of complexation.     

A versatile assay to study cellular uptake of gene transfer complexes by flow cytometry

In this study, we present a simple and reliable method to analyse the first steps of DNA-based gene delivery into eucaryotic cells, i. e. binding and internalisation of transfection complexes. Taking advantage of flow cytometry, it is possible to discriminate quantitatively between total and internal DNA on a single-cell level. Here, we use two fluorescent dyes with high specificity and affinity to double-stranded DNA that cannot penetrate the extracellular membrane of living cells. Total DNA is stained prior to complexation with the first dye and complexes are added to cells. After the incubation, only extracellular DNA remains accessible to the second dye. Cell associated fluorescence is measured simultaneously using a flow cytometer and data are analysed using a computer program capable of calculating the ratio of fluorescence intensities on a single-cell level. These ratios are indicative of the binding and internalisation kinetics of gene transfer complexes.     

Romanowsky dyes and Romanowsky-Giemsa effect. 5. Structural investigations of the purple DNA-AB-EY dye complexes of Romanowsky-Giemsa staining.

A reproducible Romanowsky-Giemsa staining (RGS) can be carried out with standardized staining solutions containing the two dyes azure B (AB) and eosin Y (EY). After staining, cell nuclei have a purple coloration generated by DNA-AB-EY complexes. The microspectra of cell nuclei have a sharp and intense absorption band at 18,100 cm-1 (552 nm), the so called Romanowsky band (RB), which is due to the EY chromophore of the dye complexes. Other absorption bands can be assigned to the DNA-bound AB cations. Artificial DNA-AB-EY complexes can be prepared outside the cell by subsequent staining of DNA with AB and EY. In the first step of our staining experiments we prepared thin films of blue DNA-AB complexes on microslides with 1:1 composition: each anionic phosphodiester residue of the nucleic acid was occupied by one AB cation. Microspectrophotometric investigations of the dye preparations demonstrated that, besides monomers and dimers, mainly higher AB aggregates are bound to DNA by electrostatic and hydrophobic interactions. These DNA-AB complexes are insoluble in water. Therefore it was possible to stain the DNA-AB films with aqueous EY solutions and also to prepare insoluble DNA-AB-EY films in the second step of the staining experiments. After the reaction with EY, thin sites within the dye preparations were purple. The microspectra of the purple spots show a strong Romanowsky band at 18,100 cm-1. Using a special technique it was possible to estimate the composition of the purple dye complexes. The ratio of the two dyes was approximately EY:AB approximately 1:3. The EY anions are mainly bound by hydrophobic interaction to the AB framework of the electrical neutral DNA-AB complexes. The EY absorption is red shifted by the interaction of EY with the AB framework of DNA-AB-EY. We suppose that this red shift is caused by a dielectric polarization of the bound EY dianions. The DNA chains in the DNA-AB complexes can mechanically be aligned in a preferred direction k. Highly oriented dye complexes prepared on microslides were birefringent and dichroic. The orientation is maintained during subsequent staining with aqueous EY solutions. In this way we also prepared highly orientated purple DNA-AB-EY complexes on microslides. The light absorption of both types of dye complexes was studied by means of a microspectrophotometer equipped with a polarizer and an analyser. The sites of best orientation within the dye preparations were selected under crossed nicols according to the quality of birefringence.(ABSTRACT TRUNCATED AT 400 WORDS)     

Arsenazo III forms 2:1 complexes with Ca and 1:1 complexes with Mg under physiological conditions. Estimates of the apparent dissociation constants.

Experiments to determine the apparent dissociation constants of the Ca and Mg complexes of arsenazo III clearly indicated that the predominant Ca complex contains one Ca ion and two dye molecules, although previous reports have either claimed or assumed 1:1 complexing. The evidence is based on the effects of varying [dye] as well as [Ca] and [Mg], and clear evidence for the formation of 1:1 complexes with Ca was obtained only at submicromolar [dye], whereas Mg formed 1:1 complexes exclusively. The implications of these findings with regard to the use of arsenazo III as an indicator of intracellular free [Ca] are discussed, with particular reference to its selectivity for Ca and the interference effects of other ions.     

Spectroscopic properties of complexes of acridine orange with glycosaminoglycans. I. Soluble complexes.

The interaction of acridine orange with dermatan and chondrotin sulfates results in the formation of complexes containing bound dye molecules ordered into dissymmetric arrays. Complexes containing an excess of available disaccharide residues compared to dye are completely soluble, and exhibit biphasic circular dichroism bands. Analysis of the dependence of the extrinsic circular dichrosim on dye aggregation indicates the presence of extended dye stacks bound to the glycosaminoglycan. Complexes formed in solutions containing an excess of dye are only partially soluble, and exhibit circular dichroism spectra having band shifts and intensity changes relative to the soluble complexes. The latter complexes show a sharp drop in induced circular dichroism with temperature, due to a cooperative change in the structure of the complex. The structural order of the dye–glycosaminoglycan complex may differ from the intrinsic structure of the glycosaminoglycan itself in dilute solution.     

Heterogeneity of binding sites of proflavin on DNA

The desorption and melting with temperature of proflavine–DNA complexes has been studied by spectrophotometry and spectrofluorometry. Two methods are described to determine at each temperature the concentration of free and bound dye. The first one is based on the quenching of fluorescence of the free dye by the iodine ion, the second on fluorescence polarization measurements. It is shown that the sites where the bound dye fluoresces are thermally less stable than those where it is quenched, in such a way that a redistribution of the dye between the two types of sites occurs at intermediate temperatures, leading to a drop in the total fluorescence. This confirms the nature of the “emitting” sites which correspond to AT-rich region, while “quenched” sites correspond to GC-rich region. The first have a larger binding constant at room temperature, but only the latter are stabilized by dye intercalation. The desorption and melting have also been followed through the relative changes of absorption. The curves obtained at different wavelengths are not superimposed which is at variance with what is observed with complexes of proflavine with poly dAT and poly dG.dC. The beginning of the desorption process corresponds to minor variations at 445 nm, the maximum of absorption of the free dye, but large changes occur at 460 nm, the maximum of the difference spectrum of the complexes proflavine–poly dAT and proflavine-poly dG.dC. The spreading of the melting curves for different wave lengths must therefore reflect the dependence of the absorption spectra of the dye on the nature of the neighboring bases. However, the action spectrum of the fluorescence, which gives the absorption spectrum of the “emitting” sites only, is identical with the total absorption spectrum of the bound dye.     

Interactions of 4'', 6-diamidine-2-phenylindole with synthetic polynucleotides.

4', 6-Diamidine-2-phenylindole forms fluorescent complexes with synthetic DNA duplexes containing AT, AU and IC base pairs; no fluorescent complexes were observed with duplexes containing GC base pairs or with duplexes containing a single AT base pair sandwiched between GC pairs. The binding site size is one molecule of dye per 3 base pairs. The intrinsic binding constants are higher for alternating sequence duplexes than for the corresponding homopolymer pairs. With the exception of the four-stranded helical poly rI which exhibits considerable fluorescence enhancement upon binding of the ligand, none of the single- or multi- stranded polyribonucleotides and ribo-deoxyribonucleotide hybrid structures form fluorescent complexes with the dye. Poly rI is the only RNA which forms a DNA B-like structure (Arnott et al. (1974) Biochem. J. 141, 537). The B conformation of the helix and the absence of guanine appear to be the major determinants of the specificity of the fluorescent binding mode of the dye. Nonfluorescent interactions of the dye with polynucleotides are nonspecific; UV absorption and circular dichroic spectra demonstrate binding to synthetic single- and double-stranded DNA and RNA analogs, including those containing GC base pairs.     

Fluorescence polarization study of DNA--proflavine complexes

The binding of proflavine to DNA has been studied by measuring the polarization and intensity of emission of DNA–dye complexes. Such measurements also permit the determination of the fluorescence of the bound dye as a function of the degree of binding. Techniques of emission spectroscopy permit the study of complexing at high phosphate to dye ratios, and we have examined complexes formed at up to 12,300:1 phosphates to dye. At high phosphate to dye ratios, we find that equilibrium plots of the binding data show only one type of binding. Reports in the literature of multiple binding constants are shown to be due to the incorrect assumption that the fluorescence of the bound dye is independent of the amount bound. The emission properties can be qualitatively accounted for by assuming that nearest-neighbor interaction between bound dyes quenches the fluorescence. We report that, within experimental error, the binding constant is insensitive to the base content of the DNA. The DNA-dye complexes show a temperature dependent depolarization, the cause of which is, as yet, unknown. Heat denaturation of the DNA–dye complex may be followed on a Perrin plot.     

A simple and sensitive method for the quantitative estimation of collagen.

A method to quantitate collagen in solution is described. It is based on binding of the dye Sirius Supra red F3BA by collagen followed by elution and estimation of the bound dye in a spectrophotometer. The assay can be used in the range from 10 to 100 μg protein. The method is rapid, specific, simple, sensitive, and highly reproducible.     

Study of the interaction of proflavin with deoxytetraribonucleoside triphosphate 5'-d(ApGpCpT) in aqueous solution by uni- and two-dimensional 1H-spectroscopy]

Complex formation between acridine dye proflavine and self-complementary deoxytetraribonucleoside triphosphate 5'-d(ApGpCpT) in water-salt solution was studied by the method of one- and two-dimensional 1H-NMR spectroscopy (500 MHz). Two-dimensional homonuclear 1H-NMR spectroscopy (2D-COSY and 2D-NOESY) was used for complete assignments of proton signals of molecules in solution and for qualitative analysis of the nature of interactions between proflavine and tetranucleotide. Concentration dependences of proton chemical shifts of the molecules were measured at 293 K. Equilibrium reaction constants and limiting chemical shifts of dye protons in the complexes were determined using suggested schemes of complex formation. Based on the obtained data possible types of complexes were considered. Analysis of relative content of different types of complexes was made and special features of dynamic equilibrium were revealed as a function of correlation of dye and tetranucleotide concentrations. The most favourable structure of 1:2 complex of dye with tetranucleotide was constructed using the calculated values of induced chemical shifts of proflavine protons and 2D-NOESY spectra.     

Quantitative determination of sulfhydryl groups with "mercurochrome" (author's transl)

Dibrommercuryfluoresceine (DBMF) reacts stoichiometrically and quantitatively with the thiol group of cysteine, glutathione and thioglycolic acid respectively, at pH 7.0. Polarographical and spectrometrical titrations clearly show that in the spectra of the investigated mercaptides the wave length of the first absorption maximum of DMBF (507 nm) remains unchanged but the molar extinction coefficient increases by approximately 20%. Serum albumin, ovalbumin, beta-lactoglobulin and glyceraldehydephosphatedihydrogenase, after incubation with DBMF, form adducts with the dye from which the pure mercaptide complexes were separated by means of column chromatogrphy. These complexes were separated by means of column chromatography. These complexes show a bathochromic shift (520 nm) of the dye band which is decreased now by 50%. The molar extinction coefficient epsilon 520 has been determined from 32,000 to 33,850. On the basis of these values SH-contents of the four proteins were obtained which are in good accordance with data previously published in the literature. No selective reaction, f.i. with more accessible or/and reactive SH-groups was observed. After 30 min incubation with DBMF and washing with isotonic phosphate buffer, native animal tumor cells show in the main absorption band the bathochromically shifted dye maximum. A first temptative estimation of the protein SH-groups yielded 1.7-2.1 X 10(-14) mole SH/single cell. This result lies between the SH-content determined microspectrometrically on cells stained with DDD-Fast Blue B (1.1-1.55 X 10(-14)) and macroscopically on cell homogenates with DTNB (3.1 X 10(-14)). Up to now, no certain information can be given whether or to what extent unspecific absorption effects possibly might be involved in the data obtained with DBMF treated cells, but interaction with nucleic acids can be excluded with certainty on the basis of relevant model experiments.     

Interaction between transcobalamin II and immobilized Cibacron Blue F3GA

Human serum transcobalamin II (TC II), a vitamin B₁₂ (Cbl) transport protein, complexes with Cibacron Blue F3GA, a reactive blue dye which can bind to proteins that require nucleotides as cofactors. Apo-TC II and holo-TC II both bind, but intrinsic factor (IF) and R-type binders of Cbl do not. Other mammalian species TC II also complex with the dye. Greater than 87% of the applied TC II-CN-[⁵⁷Co]Cbl remains bound to the dye even at pH 4.0. At pH values below this, the CN-[⁵⁷Co]Cbl dissociates off TC II which remains bound to the dye. High salt concentrations will break the TC II-dye complex. Ionic forces were considered not to be involved since complexing also occurred at pH 9.0, 2.5 pH units above the isoelectric point of TC II. Failure to dissociate the TC II-dye complex with 50% glycerol makes hydrophobic interactions unlikely. In addition to the potential uses of TC II-Cibacron Blue F3GA complexes in a total scheme for protein purification, the possibility that TC II is a nucleotide-requiring protein should be explored.